Collection of the Plant: Gum nodules were collected and authenticated at the Herbarium of Medicinal and Aromatic Plants & Traditional Medicine Research Institute with code number G-1983-1- MAPTRI-H. After that, nodules were dried on air at room temperature away from the sun and processed in two different processing methods:
- Spray drying technique.
- Manual grinding with a pestle and mortar.
Three Gum Arabic samples were sent to the National Public Health Laboratory to ensure that they met microbiological standards. These samples included the spray-dried GA, the manually ground GA (with a pestle and mortar), and ready-made mechanically ground powder from a private company.
The analysis showed that the ready-made mechanically ground sample was contaminated with E-coli. Moreover, Yeasts and Molds have also been detected with levels exceeding the acceptable limits in this sample. The other two samples were matched with the Microbiological Standard for Gum Arabic. However, the spray-dried GA showed better results than the manually ground sample. These two samples were the samples used in this study.
Preparation of extract: For each type of Gum Arabic, three samples of 50-gram weight were extracted with ethanol of different concentrations - 70%, 80%, and 99.9%. The samples were soaked in different concentrations of ethanol (Reagents Duksan, Ethyl Alcohol, absolute, product No.6923, 2.5L)and then left for three days while being filtered daily. Afterward, the solvents were evaporated, and the extracts were freeze-dried until they were completely dry. The yield percentages of the three ethanolic extracts (70%, 80%, 99.9%) of both types of Gum Arabic were compared.
Antibacterial susceptibility test: Antibacterial susceptibility tests were conducted for the 70% ethanolic extract of both types of Gum Arabic against Enterococcus faecalis using the Agar disc diffusion method [17-19], with the following steps:
1. Preparation of the media: Muller-Hinton Agar (HIMEDIA, REF. M173-500g Mueller Hinton Agar) was prepared according to the instructions. Each 20 mL of the freshly prepared and autoclaved Muller-Hinton agar was poured into a sterile petri dish and maintained at room temperature to cool down. Before use, the plates were incubated at 35 °C for 48 hours, and the sterility was checked.
2. Preparation of discs: Filter paper discs of 6 mm diameter were prepared from Whatman filter paper No. 1, placed in a petri dish, and sterilized in a hot air oven at 160 °C for two hours.
3. Preparation of extracts solutions: The extract from each type of Gum (200mg) was dissolved in 1ml of 20% ethanol (Reagents Duksan, Ethyl Alcohol, absolute, product No.6923, 2.5L). The mixture was mixed well using a vortex to ensure complete dissolution.
To avoid synergistic effect of the solvent (ethanol 20%) on the antibacterial activity of the extract:
- Checking the inhibition zone of ethanol 20%: A disc impregnated with ethanol 20% was placed on a plate inoculated with E. faecalis and incubated at 37°C for 24 hours under anaerobic conditions. The solvent was used after no inhibition zone was observed around the disc.
- Selection of the negative control: Ethanol 20% itself served as a negative control. According to the protocol of disc diffusion technique, the solution that used to dissolve the tested material should be used as a negative control [18, 19].
4. Reactivation and inoculation of Enterococcus faecalis: To reactivate the standard strain Enterococcus faecalis (ATCC 29212), it was placed in 5ml of brain heart infusion (BHI) broth (HIMEDIA, REF. M210-500G Brain Heart Infusion Broth) and kept under anaerobic conditions for 48 hours. Next, a sterile cotton swab was used to transfer E. faecalis from the BHI broth onto a Muller Hinton Agar plate. The plate was then incubated at 37 degrees Celsius under anaerobic conditions for 24 hours.
5. Preparation and standardization of inoculum suspension: Three to five well-isolated colonies of the same morphological type were selected from a Muller Hinton Agar plate culture and transferred with a sterile loop into a tube containing 5 ml of BHI broth. The broth was incubated anaerobically at 37 °C for 24 hours.
The turbidity of E. faecalis suspension was adjusted equal to that of 0.5 McFarland standard (1.5 × 108 CFU/ml) by using BHI broth for
dilution of E. faecalis suspension.
6. Antibacterial susceptibility test: Plates inoculated with Enterococcus faecalis were prepared by streaking the surface of Muller Hilton Agar plates with the adjusted bacterial suspension using a sterile cotton swab.
Three sterile discs were immersed in 10 µL of both extract solutions of Spray-dried Gum Arabic and mechanically ground Gum Arabic. After saturating the discs, they were moved to prepared plates.
Discs were also immersed in Ethanol (20%) to be used as a negative control, while Chlorhexidine 0.2% (Clenora mouthwash, Chlorhexidine Gluconate BP 0.2%w/v) as well as Sodium hypochlorite 1% (Prevest Denpro Hyposol, 3% Sodium hypochlorite 500 ml) to be used as positive controls transferred to the prepared plates.
Ready-made antibiotic multidisc for Gram-positive isolates (Axiom laboratories, Code No. 001, Mantola Pahar Ganj- New Delhi-110055) was transferred to the prepared plate. The antibiotics included in the multidisc for Gram-positive were displayed in (Figure 1).
These plates were incubated at 37°C for 24 hours under anaerobic conditions. The plates were observed for the emergence of a clear zone around each disc (inhibition zone). The diameters of the inhibition zones were recorded.
7. Antibacterial susceptibility test for serial dilutions of Gum Arabic (100, 50, 25, and 12.5 mg/ml) against Enterococcus faecalis:
Both types of Gum Arabic were coded and diluted in four serial dilutions to obtain concentrations of 100, 50, 25, and 12.5 mg/ml.
Two Muller-Hinton Agar plates were prepared, and labeled according to the extract codes. Each plate was then divided into four labeled zones indicating the extract concentrations. Sterile cotton swab was rolled in the Enterococcus faecalis suspension to streak the surface of Muller Hinton Agar.
For both types of Gum Arabic, four sterile discs were immersed in 10 µL of each concentration (100, 50, 25, 12.5 mg/ml). The discs were transferred to their labeled zones in the coded plates. The plates were incubated at 37°C for 24 hours under an anaerobic condition, which was generated by a CO2 generating kit (Thermo Scientific, AnaeroJar™ 2.5L with Oxoid CO2 Gen Sachet). After incubation, the diameter of the inhibition zones were measured.
An excel plot to display the relation between the concentration of Gum Arabic and the inhibitory effect was conducted for each type of Gum Arabic. The square of the radii diameter of the inhibition zone was presented on the Y-axis, and the log concentrations of Gum Arabic on the X-axis. A suitable curve was drawn from the plot for each type of Gum Arabic. Simple regression analysis was performed to determine the relation between the concentration of Gum Arabic and its inhibitory effect as well as the concentration of each type of Gum Arabic which shows no inhibition [20].
Radius inhibition zone equal ½ [Diameter of inhibition zone in millimeter including disc diameter ̶ disc diameter] [20]